Column chromatography has long been known as an accepted analytical method by which separation of the solutes of a liquid sample is made possible. A recent development known as High Performance Liquid Chromatography (H.P.L.C.) has introduced a new concept of separating the solutes of the sample with high speed and with near predictable performance.
As in all chromatography, separation of the solutes of the sample using `Reversed-Phase` (RP) is accomplished with high speed; in a fraction of the time usually required; with the capability of separating polar/non-polar ionic solutes many times on a single column using a simple mobile phase. Also, the reversed-phase columns, with proper use, can give long service.
The term `Reverse-Phase` was referred to by Howard et al (Biochem. J., vol. 46, 1950). The `Reversed` term is pertinent since in `Normal` phase partition chromatography the stationary phase is polar and the mobile phase is non-polar, while in the `Reversed-Phase` chromatography, the stationary phase is non-polar and the mobile phase is polar. Thus, the polarity relationship is reversed. The mechanism assumed to account for this is the binding of the solutes to the functional groups of the hydrocarbon chains at the surface of the stationary phase in such a way that the binding forces vary in strength depending upon the nature of the solute-functional group interaction. The most commercial bonded `Reversed-phase` media, silica gel is reacted with an organic chlorosilane, i.e. the hydrocarbon moiety is covalently bonded to the silica surface as Si--O--Si--R, where R=alkyl chain, the most common being C.sub.18.
All `Reversed-phase` HPLC does not use the C.sub.18 chain. Scott et al (J. Chromatog., vol. 142, 1977) pointed out that packing media having carbon chains of the same length can be very different chromatographically. In general, the solute retention is directly proportional to the chain length of the packing media i.e. the retentive capacity is greater for longer chains and also the column stability is greater for longer chain phases (Majors et al., J. Chromatog., vol. 12, 1974; and Kingston et al., J. Chromatog., vol. 104, 1975; and vol. 116, 1976). Also, the retentive capacity depends on the carbon content of the `Reversed-phase` packing media. The carbon content is the percentage of carbon (w/w) of the alkyl chain containing silica, e.g. octadecyl-silica, based on the weight of the alkyl chain. The carbon content refers to the interacting functional groups, the alkyl chains. The unreacted hydroxyl groups give the stationary phase polar characteristics and therefore selectively depends in part upon the extent of silanization. The carbon content is, for chromatographic purposes, distinguished from derivatization (surface coverage, % silanization). Derivatization is defined as the percentage of the available hydroxyl groups of the silica gel reacted with the silane.
The procedure described by Kingston et al. (J. Chromatog., vol. 116, 1976) provides Silica-C.sub.18 having 11.21 percent carbon content.